Cancer is caused by cumulative multiple genetic mutations, which result in the activation of oncogenes and/or the inactivation of tumor suppressor genes. Cancer remains a major cause of mortality worldwide. Despite advancements in diagnosis and treatment, the overall survival rate has not improved significantly in the past several years. There remains an unfulfilled need for more accurate detection and sensitive means of diagnosis of tumors.
Most cancer deaths are not caused by the primary tumor. Instead, death results from metastases, i.e., multiple widespread tumor colonies established by malignant cells that detach themselves from the site of the original tumor and travel through the body, often to distant sites. If a primary tumor is detected early enough, surgery, radiation, chemotherapy, or some combination of these treatments can often eliminate it. Unfortunately, the metastatic colonies are harder to detect and eliminate and it is often impossible to treat all of them successfully. Therefore, from a clinical point of view, metastasis can be considered as the conclusive event in the natural progression of cancer. Moreover, the ability to metastasize is the property that uniquely characterizes a malignant tumor. Cancer metastasis comprises the following complex series of sequential events: 1. Extension from the primary locus into surrounding tissues; 2. Penetration into body cavities and vessels; 3. Release of tumor cells for transport through the circulatory system to distant sites; 4. Re-invasion of tissue at the site of arrest; and 5. Adaptation to the new environment so as to promote tumor cell survival, vascularization, and tumor growth. Due to the complexity of cancer and cancer metastasis, and frustration resulting from the lack of effective treatment for late-stage cancer patients, much effort has been invested in developing tests to detect development of metastasis and early relapse.
Circulating tumor cells (CTCs) are cancer cells that are shed from either the primary tumor or its metastases and that circulate in the peripheral blood. While metastases are directly responsible for the majority of cancer deaths, CTCs may constitute seeds for metastases and may indicate the spread of the disease. The ability to identify CTCs when they are very sparse (at most a few CTCs per ml) could allow early detection of indications of a cancer, or even of a precancerous growth before the appearance of evident clinical symptoms. Potential interest in detection of CTCs in peripheral blood was first suggested over a century ago, but then subsided because they are difficult to detect by conventional methods due to low numbers of CTCs in a sample. The challenge is to develop an approach that is capable of both highly sensitive and highly specific identification and characterization of rare tumor cells circulating in the blood, enabling such cells to be distinguished from normal epithelial cells and leukocytes. Detection of circulating tumor cells could facilitate cancer prognosis, diagnosis of minimal residual disease, assessment of tumor sensitivity to anticancer drugs, and personalization of anticancer therapy. Highly sensitive and specific identification of CTCs would also have potential application in early diagnosis and screening of invasive cancers.
Molecular techniques based on PCR amplification of tumor-specific abnormalities in DNA or RNA have facilitated detection of occult (hidden) tumor cells. PCR-based tests capable of routinely detecting one tumor cell in one million normal cells have been devised for identification of circulating tumor cells in various types of carcinomas. For example, Smith B. et al. develops reverse transcriptase-polymerase chain reaction to detect melanoma cells in peripheral blood (Lancet, 338: 1227-1236, 1991). However, these methods may not effectively distinguish viable tumor cells from normal cells. Since cell integrity is destroyed during DNA or RNA extraction, this approach precludes the analysis of cell morphology and phenotype, and so may be unable to distinguish material shed directly from normal tissue as opposed to from tumors, nor allow detection of several associated changes in the same cell.
Immunofluorescence microscopy enables analysis of cell morphology and direct counting of identifiable presumptive tumor cells. Detection is carried out by immunolabeling of cells using appropriate antibodies. However, there are so far no antibodies for tumor specific antigens used to identify CTCs. In addition, immunomagnetic cell separation with immunocytochemical labeling has been developed and evaluated for detection of CTC clusters in colorectal carcinoma patients (Clinical Cancer Research, 2001, Vol. 7, pp. 4080-4085). WO 2006050352 provides an improved cell adhesion matrix (“CAM”) and an improved cell isolation device for separating target cells such as tumor, fetal and angiogenic cells from blood or other fluid tissue samples. Furthermore, WO 2009051734 discloses a device for capturing circulating, nonhemopoietic tumor cells. The device includes a microfluidic channel to which is bound a tumor specific binding agent; and a pump producing a continuous, unidirectional shear stress of 0.1 to 20 dyn/cm2 in the channel. However, the above techniques cannot effectively detect CTC.
It is apparent that there is need for a method and/or kit for identifying cells in circulation having metastatic potential prior to establishment of a secondary tumor, particularly during the early stages of cancer.